Numerous medical conditions require treatment via injection of gamma globulin. The manner of preparation of the gamma globulin is of critical importance, particularly in order to eliminate the chance of contracting viral hepatitis. Viral hepatitis is a debilitating disease at best and lethal at worst. Consequently, any advances made to eliminate the chance of contamination of any injectable product by this virus are of immense importance.
Hepatitis B virus is estimated to infect approximately 200 million persons worldwide. Since the base material for the production of gamma globulins often is plasma obtained from a human source, the chances of obtaining contaminated or infected plasma are significant in view of the substantial number of persons who are chronically and acutely infected. The infected plasma from such a person may contain not only varying amounts of viral particles but also different sizes and forms of the particles. The most common form is the spherical particle which has a mean diameter of 22 nm. These spherical particles are devoid of DNA and represent free envelopes of the virus. Less common are the 42 nm Dane particles which represent the virion and consist of an envelope and a 27 nm nucleocapsid that contains a molecule of DNA. Free nucleocapsids may be observed in the nucleus of infected hepatocytes but are generally not found in the plasma. Infected hepatocytes have been found to synthesize excessive amounts of envelope which can be found circulating throughout the body. In accordance with the different components of viral particles, different immunological markers have been identified. For example, associated with the core is an antigen commonly labelled HB.sub.c Ag and an "e" antigen labelled HB.sub.e Ag. The most common antigen employed for the detection of hepatitis B virus infection, however, is the surface antigen HB.sub.s Ag. The structure and genetic organization of the hepatitis viral particle has been reviewed in an article by Tiollais et al., in Biology of Hepatitis B Virus, Science, Vol. 213, 406-411 (July, 1981). Further discussion concerning the association of the Australian antigen with persistent or chronic hepatitis may be obtained in Australia Antigen and Hepatitis by Blumberg et al., C.R.C. Monotopic Series, C.R.C. Press, Cleveland, Oh. (1982). The close relationship of hepatitis B surface antigen to hepatitis viral infectivity is discussed by Blumberg, supra, on page 14.
Historically, a number of tests have been developed for the testing and identification of hepatitis-type viral infections and are uniformly directed towards the detection of hepatitis related antigens or the antibodies specific therefor. These tests have generally been characterized as a first, second or third generation test depending upon their sensitivity in the detection of weakly positive HB.sub.s Ag reference panel samples obtainable from the U.S. Bureau of Biologics. Presently, the most sensitive tests available are of the third generation category and include radioimmunoassay, enzyme linked immunosorbent assay, reversed passive hemagglutination and reversed passive latex agglutination tests. Typical third generation immunoassays for HB.sub.s Ag will detect approximately 10.sup.9 particles per ml of serum. Unfortunately, the primary particles to be detected are noninfectious 22 nm spherical forms. It is known that there is significant variation in the number of infectious Dane particles and noninfectious structures between sera. Therefore, sera can be diluted significantly past the point where they are positive in an immunoassay and still be infectious. See the article entitled "Hepatitis B virus infection in Chimpanzees, Titration of subtypes." in J. Infec. Dis. 132:451-459 (1975) by Barker et al. It thus becomes readily apparent that even a negative result with the most sensitive test available will fail to ensure the noninfectivity of a sample. Consequently, the manner of preparation for an immunoglobulin injectable reagent from a potentially infected plasma source becomes of paramount importance since any production method should ideally be capable of removing or destroying substantially all infective viral particles. At present, only in vivo chimpanzee studies are sufficiently sensitive to ensure noninfectivity of any particular sample. The cost and the requirements of such studies make them prohibitive for routine use. See "The Test for HB-Associated Antigens and Antibodies" by Gerety et al. in Chapter 11 of Viral Hepatitis, Ed. Vyas et al., The Franklin Institute Press, Philadelphia, Pa. (1978).
Presently, all immunoglobulin injectable materials approved for use by the FDA and Bureau of Biologics have been produced by the alcohol fractionation procedure developed by Dr. E. Cohn of Harvard during the 1940s and described in Cohn et al., J. Am. Chem. Sos., 68, 459 (1946). This procedure, coupled with the careful selection of plasma negative for hepatitis infectivity, determined by the most sensitive tests available, has been employed for such a long period of time (i.e., since the 1970's) that the U.S. government has adopted a position favoring only the resultant preparations of this procedure as safe. That the products produced by this procedure are indeed safe can easily be demonstrated by the millions of non-infected recipients of product. Unfortunately, occasional problems still arise demonstrating that despite the favorable appearance of the `numbers` correlated with the Cohn process, the Cohn process still will not ensure complete noninfectivity. Despite the apparent success of the Cohn process to produce a safe human gamma globulin product, many investigators have attempted to find a replacement method which would involve fewer steps and milder conditions, offer higher yields and eliminate the presence of aggregates. The presence of the latter limits the current product to use for intramuscular injections only. The Cohn process is also disadvantageous because vast volumes of plasma are required due to the inherent low yield. Plasma is not only expensive but is also present only in limited supply.
It is an object of the present invention to provide a process whereby hepatitis B virus infectivity may be safely eliminated from a plasma by a more efficient process than that developed by Cohn et al. The resulting gamma globulin product would also be safe for both intramuscular or intravenous injections.
Several conventional methods for the separation of gamma globulin from human serum have been described notably by Baumstark et al. in "A Preparative Method For The Separation Of 7S Gamma Globulin From Human Serum", Archives of Biochemistry and Biophysics, 108, 514-522 (1964) and by A. J. Webb in "A 30-Minute Preparative Method For Isolation Of IgG From Human Serum", Vox Sang, 23:279-290 (1972). Although both of these papers are more concerned with the separation and selection of various gamma globulin classes from a serum containing numerous other contaminating proteins, they do address the removal of contaminating proteins and materials from the original serum sample. Both employ a DEAE-Sephadex column chromatographic material with a phosphate buffer eluting agent. Both investigators met with some degree of success as far as removal of contaminating proteins was concerned. However, both failed to address the problem of removing contaminating hepatitis viral particles in order to provide a safe, injectable reagent.
It is yet another object of the present invention to provide methods utilizing double chromatographic column resin/buffer combinations which are more effective in removing contaminating hepatitis viral particles than those provided by conventional methods.
Another method, described by Stanworth in an article entitled "A Rapid Method Of Preparing Pure Serum Gamma Globulin", Nature, 188, 156-157 (1960), involves the use of a diethyl amino ethyl cellulose anion exchanger to remove proteins from human serum dialyzed to remove high molecular weight proteins, however, the method described fails to account for the effect on hepatitis viral contaminants and additionally fails to provide an injectable reagent, both of which are objects of the present invention.
Condie has described in U.S. Pat. No. 4,136,094, "Preparation of Intravenous Human and Animal Gamma Globulins And Isolation Of Albumin", another method for obtaining gamma globulin which is claimed safe for intravenous administration. Condie's method involves three manipulations including plasma stabilization by treatment with fumed colloidal silica, isolation and elution of gamma globuin and albumin from ion exchange resins and finally concentration dialysis and sterile filtration. The fumed colloidal silica step is provided to remove hepatitis associated antigen present in the plasma as well as a number of proteolytic enzymes and their precursors. The colloidal silica treated materials were tested for presence of hepatitis associated antigen by radioimmunoassay. The materials tested negative and intravenous administration of large quantities (in excess of 30 g) in over 50 patients showed no evidence of passage of hepatitis virus nor produced cases of hepatitis. To be noted, however, as previously discussed, testing by presently available radioimmunoassay procedures will not ensure that the tested sample is free of infective hepatitis. Without further testing, any such material will not be approved by the U.S. government for widespread use in excess of that required for limited clinical studies. It is also to be noted that in the Condie process, there is a potential health hazard to workers due to exposure to silica fumes. The present invention avoids such exposure.
It is still another object of the present invention to provide a significantly simpler, effective procedure for isolating immunoglobulins from blood plasma and hepatitis associated antigens which does not require fumed colloidal silica.
Treatment of hemolytic disease of the fetus or newborn has become rather standard and is accomplished by treatment of the mother by injection of Rho (D) immunoglobulin of human origin. Such a product is RhoGAM, available from the assignee hereof, operates by preventing the unimmunized Rho (D) negative mother from responding to Rho (D) antigen present on red cells and `received` at delivery from an Rho (D) positive infant. Thus, by preventing anti-Rho (anti-D) production by the mother at delivery, the subsequent Rho (D) positive infant of this mother is protected from hemolytic disease of the newborn. Although this successful product is presently produced by a Cohn alcohol fractionation type process, several investigators have attempted to use alternative methods to produce similar materials to thereby provide an economically more advantageous product and to reduce large plasma requirements. Such investigational efforts have been reported by Hoppe et al. in "Prevention of Rh Immunization Modified Production of IgG Anti-Rh For Intravenous Application By Ion Exchange Chromatography", Vox Sang, 25:308-316 (1973), Friesen et al. in "Column Ion-Exchange Preparation and Characterization of an Rh Immune Globulin for Intravenous Use", Journal of Applied Biochemistry, 3, 164-175 (1981), and Walsh, T. J. and O'Riordan, J. P. in "A review of the production and clinical use of intravenous Anti-D immunoglobulin", Irish Med. J. 75, 232-244 (1982).
Hoppe in Germany, Friesen in Canada and O'Riordan in Ireland, all employed a DEAE-Sephadex chromatography column in conjunction with a phosphate buffer eluding agent. Hoppe's source of anti-D containing plasma was from volunteers who passed an HB.sub.s Ag laboratory test for at least six months, the plasma being stored in the interim. Thus, Hoppe employed a relatively safe, noninfective plasma to start with. Some screening work was done in Hoppe's laboratory to confirm the affinity of HB.sub.s AG for these resins. To our knowledge, no animal safety work was done to confirm removal of infectivity. Hoppe's concern was directed towards the removal of aggregated materials and the isolation of an unfragmented, immunoelectrophoretically pure IgG having a relatively high antibody concentration. The Freisen publication reports on the modifications made to the Hoppe method for the development of an intravenous Rh IgG for use in Canada. As Hoppe had done, Freisen tested each unit of Rh plasma for HB.sub.s AG to eliminate any donors testing positive. Freisen employed the radioimmunoassay kit from Abbott Laboratories, North Chicago, Ill. (Ausria II Kit). This test is still regarded as one of the most sensitive and was also employed in the development of the invention described later. Freisen reported that clinical trials showed the material produced using the DEAE-Sephadex resin/phosphate buffer combination was effective and safe for the prevention of Rh immunization. Freisen, however, reported no additional tests for determining the efficacy of the DEAE-Sephadex/phosphate buffer combination for removing hepatitis B virus infectivity from plasma samples. This, at least from the U.S. government's perspective, is especially important since the radioimmunoassay test employed in screening the donor plasma samples is incapable of detecting concentrations of HB.sub.s AG particles two or three orders of magnitude lower which may still be infective. It is this concern for the potential infectivity of a reagent produced by such a method that the U.S. government has been significantly more restrictive in permitting the production of injectable immunoglobulin reagents by ion-exchange methodologies.
It is an object of the present invention to provide resin/buffer systems that are superior in their ability to eliminate hepatitis B virus infectivity than those employed by previous investigators.
Zolton et al. in U.S. Pat. No. 4,434,093 have described methods for producing from human serum, human gamma globulin essentially free of HB.sub.s Ag. Specifically provided were single column "soft" ion exchange A-50 type resin/buffer systems capable of effectively removing HB.sub.s Ag thought to be closely corrolated with viral hepatitis type B infectivity. However, the "soft" resins used in U.S. Pat. No. 4,434,093 are not convenient for handling large volumes of plasma, thus creating "scale-up" difficulties. Furthermore, in view of the fact that only a single column was used in order to effectively remove HB.sub.s Ag, the ratio of A-50 resin per ml of applied sample was required to be no lower than 160 mg/ml to insure adequate removal of surface antigen. The resin used is extremely expensive and this is compounded by the fact that, for safety reasons, such resin should preferably be virgin and discarded after each trial.
It is an object of the present invention to provide double column resin/buffer systems which are better adapted for large-scale operation.